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Determining the size of fluorescence spots in biological images 10 times below the diffraction resolution limit.
Despite its popularity, LSM is has a limitation, due to the diffraction limit of visible light; in the image, spots appearing as having a diameter of ~250 nm may in reality be anywhere from 250 nm down to ~2 nm. Since a commonly investigated feature in cells is the clustering, or aggregation, of protein molecules, the diffraction limit imposes a serious limitation: Aggregates in the cell membrane may for example have a physical size of 20 nm and contain ~300 protein molecules, or they may be 200 nm large and contain as many as ~30 000 protein molecules. Still, these two spots will appear to be equally large in an LSM image.
Some indication about the number of protein molecules in a spot may be given by the brightness of the spot, but this indication is known to be uncertain, because fluorophores quench each other when being close, and several fluorophores bound together may have the same brightness as a single one.
The idea in this task is a very promising one: By cross-correlating the signals from different labeled parts on the cell membrane, information will be obtained about the physical size of objects down to ~25 nm in size. Thus, compared to the present capability of LSMs, the size of objects of 10 times smaller diameter, corresponding to 100 times smaller area, will be determined. However, no change in the LSM-instrumentation is required.
The same cross-correlation approach can also be applied to images taken with a so-called STED-microscope, which we have in our lab. STED- microscopes have a resolution down to ~40 nm, but are far less commonly used than normal LSMs. If applied to STED-images, the cross-correlation approach should be able to determine the size of ~3 nm spots, corresponding to the size of individual protein molecules!
The approach has the potential to become very useful for all types of biological imaging, in academic research and in pharma industry.
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